Project Summary/Abstract: Despite advances in treatment strategies, the five year survival rate for metastatic breast cancer is ~20%. This poor prognosis is largely due to widespread metastasis and drug resistance that arises after chemotherapy. A subpopulation of tumor cells, described as ?cancer stem cells (CSC)? with mesenchymal properties are a major cause of cancer recurrence after therapy. Classical cancer drugs are poor at targeting CSCs. New drugs with novel mechanisms of action targeted to specific molecular markers and effective on mesenchymal or CSCs are therefore required. We have developed a new class of small molecule anticancer agents (referred to as CETZOLEs) that kill several breast cancer cell lines. Several additional cell lines are ~100 times less sensitive. The high degree of selectivity suggests that these compounds are not general toxins, but instead rely on certain biochemical or genetic programs in particular cells for sensitivity. CETZOLEs harness reactive oxygen species in an iron-dependent manner to kill cancer cells. Lethality is enhanced by activation of the RAS/MAPK signaling pathway. These features indicate that CETZOLEs kill cells by a caspase-independent mechanism called ferroptosis. Importantly, these compounds preferentially kill cancer cells with mesenchymal and CSC characteristics. We propose three specific aims to investigate the mechanism of action of these compounds and to begin to test their therapeutic potential against breast cancer. AIM 1. Identify the molecular target of CETZOLEs. CETZOLEs appear to induce ferroptosis by blocking cystine import through the xc- amino acid transporter, which we will test directly. To pinpoint the molecular targets, we will prepare probe molecules with photoreactive groups for photo affinity labeling of target protein(s) and biorthogonal handles for conjugation of reporter tags. Tagged compounds will be used to isolate associated proteins which will be identified by mass spectrometry. AIM 2. Relationship between EMT and ferroptosis induced by CETZOLEs. CETZOLE sensitivity is highest in cancer cells that have undergone epithelial to mesenchyme transition (EMT). Sensitivity is also modulated by E-cadherin, although the mechanism is not known. Proteins downstream of E-cadherin including Snail will be analyzed to determine how mesenchymal cells are sensitized to this class of compounds. We will test the hypothesis that Snail regulates the expression of proteins involved in cysteine biosynthesis and that mesenchymal cells are more dependent on external sources of this amino acid. AIM 3. Targeting mesenchymal breast cancer cells with CETZOLEs. Claudin-low subset of breast cancers (CLBC) represent ~15% of all cases and represent a mesenchymal form of breast cancer that we predict will be highly sensitive to CETZOLEs. We plan to analyze CETZOLE sensitivity in a panel of CLBC cell lines in vitro. Breast CSCs can arise via EMT in epithelial breast cancer. We will test whether breast CSCs are preferentially sensitive to the compounds. Efficacy of CETZOLEs in an animal model of metastatic breast cancer will be tested to begin to investigate the therapeutic potential of these novel compounds.